The proposed research is designed to investigate the molecular basis of X chromosome inactivation. X inactivation is a developmentally regulated process of control of gene expression which affects a large number of genes on the same chromosome at a precise time in embryogenesis. By using cellular systems in which the state of activity of an X chromosome can be turned on and turned off in vitro, and specific recombinant DNA generated probes, we hope to identify changes in DNA or chromatin structure which account for the phenomenae which have been observed regarding X inactivation for more than 20 years. We will utilize a cloned hypoxanthine guanine phosphoribosyl transferase probe as well as other expressed X chromosome probes generated in our laboratory to examine the molecular events attendant to X inactivation of reactivation. We will specifically examine gross genomic organization, DNA methylation, DNase I sensitivity and cell cycle timing of gene replication as a function of the state of activity of the X chronosome. Our approach is strengthened by the ability to monitor the same X chromosome against the same genetic background in both an active and inactive state. Since sex-linked diseases and X chromosome aneuploid states make up a substantial portion of the human genetic disease burden, the results of these studies will have substantial practical importance. Furthermore, these experiments should provide useful new information into the control of gene expression with attendant implications for developmental biology, carcinogenesis, etc.